Project Summary Worms (Caenorhabditis elegans) and zebrafish (Danio rerio) are premier animal models that have provided profound insight into metabolic processes related to disease. As such, a major fraction of worm and zebrafish research focuses on metabolism. Strikingly, however, <1% of these publications use metabolomics. The application of metabolomics by worm and zebrafish workers has been limited by the challenges of data interpretation. In a typical metabolomic analysis, there are thousands of signals that cannot be identified with standard experimental workflows. We have determined that this is largely because of artifacts, contaminants, and redundancies of a single metabolite. We have developed strategies to filter out these non-biologically relevant signals, but they are time intensive and require informatic expertise as well as expensive isotopic labels. With funding from the parent award, we will apply our techniques to worm and zebrafish to identify real, non-redundant metabolites. These compounds will establish reference metabolomes, which can then be specifically targeted in future experiments without the burden of untargeted data analysis. By applying our resource, the parent award focuses on the following questions: How do metabolites change with development? How do metabolites change between pigment cells within an animal? How do metabolites change with stress? We propose to use supplement funds to enhance the overall impact of the parent project by integrating two new mass spectrometry technologies: ion mobility and metabolite imaging. Ion mobility will increase the molecular resolution of our resource by providing an orthogonal measurement for metabolite identification. In addition to improving rigor, we have also found that ion mobility simplifies our credentialing technique for filtering artifacts and contaminants. In a conventional liquid chromatography/mass spectrometry analysis, credentialing necessitates that two mixing ratios of 13C-labels be used. With the extra dimension of separation resulting from ion mobility, however, credentialing can be achieved with only a single mixing ratio of 13C-labels. This reduces the amount of sample required for the project by a factor of two and will compensate for an unexpectedly low yield in generating labeled animal material. The second technology we propose to add to the project, metabolite imaging, will significantly boost impact by upgrading the output of the project to an anatomical map. Instead of reporting a simple list of metabolites present in homogenized worms and zebrafish, our resource will include a three-dimensional atlas of metabolite concentrations organized by spatial location. The proposed additions will not affect the scope of the project. The same samples will be evaluated exactly as originally described, except now the quality of data will be greatly improved with ion mobility and metabolite imaging. With the increased resolution of our resource, we will transition from answering the questions above in the context of whole worms and zebrafish to specific cells and tissues. This will support a growing interest in the community for the metabolic roles of specific cells and tissues at the organismal scale.